lable at ScienceDirect

Atherosclerosis 232 (2014) 346e360

Contents lists avai

Atherosclerosis

journal homepage: www.elsevier .com/locate/atherosclerosis

Review

Plant sterols and plant stanols in the management of dyslipidaemiaand prevention of cardiovascular diseaseqq

Helena Gylling a,1, Jogchum Plat b,1, Stephen Turley c,1, Henry N. Ginsberg d,*,1,Lars Ellegård e, Wendy Jessup f, Peter J. Jones g, Dieter Lütjohann h, Winfried Maerz i, j,k,Luis Masana l, Günther Silbernagel m, Bart Staels n, Jan Borén o, Alberico L. Catapano p,q,Guy De Backer r, John Deanfield s, Olivier S. Descamps t, Petri T. Kovanen u,Gabriele Riccardi v, Lale Tokgözogluw, M. John Chapman x,y, for the EuropeanAtherosclerosis Society Consensus Panel on Phytosterols2

aDepartment of Medicine, Division of Internal Medicine, University of Helsinki, Biomedicum Helsinki, FinlandbDepartment of Human Biology, Maastricht University, The NetherlandscDepartment of Internal Medicine, UT Southwestern Medical Center, Dallas, USAd Irving Institute for Clinical and Translational Research, Columbia University, New York, USAeDepartment of Internal Medicine and Clinical Nutrition, Sahlgrenska Academy at University of Gothenburg, SwedenfANZAC Research Institute, Concord Hospital, Sydney, AustraliagRichardson Centre for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg, Canadah Institute of Clinical Chemistry and Clinical Pharmacology, University Clinics Bonn, Germanyi Synlab Academy, Synlab LLC, Mannheim Center of Laboratory Diagnostics, Heidelberg, GermanyjMannheim Institute of Public Health, Social and Preventive Medicine, Medical Faculty Mannheim, University of Heidelberg, GermanykClinical Institute of Medical and Chemical Laboratory Diagnostics, Medical University of Graz, AustrialVascular Medicine and Metabolism Unit, University Hospital Sant Joan, IISPV, CIBERDEM, Rovira and Virgili University, Reus, SpainmDepartment of Angiology, Swiss Cardiovascular Center, Inselspital, University of Bern, Switzerlandn Faculty of Pharmacy, University of Lille 2, Lille, Franceo Strategic Research Center, Sahlgrenska Center for Cardiovascular and Metabolic Research (CMR), University of Gothenburg, SwedenpDepartment of Pharmacological and Biomolecular Sciences, University of Milan, ItalyqMultimedica IRCSS Milano, ItalyrDepartment of Public Health, University Hospital Ghent, Ghent, BelgiumsNational Centre for Cardiovascular Prevention and Outcomes, University College London, UKt Lipid Clinic, Hopital de Jolimont, Haine-Saint-Paul, BelgiumuWihuri Research Institute, Helsinki, FinlandvDepartment of Clinical and Experimental Medicine, Federico II University, Naples, ItalywDepartment of Cardiology, Hacettepe University, Ankara, TurkeyxUniversity of Pierre and Marie Curie, Paris, FranceyDyslipidaemia and Atherosclerosis Research Unit, INSERM U939, Pitié-Salpetriere University Hospital, Paris, France

qq This European Atherosclerosis Society (EAS) Consensus Panel dedicates this Position paper to the memory of Professor Tatu Miettinen, a pioneer in the study of the impactof plant sterols/stanols on cholesterol absorption and homeostasis.* Corresponding author. Irving Professor of Medicine and Director, Irving Institute for Clinical and Translational Research Columbia University, 622 West 168 Street, PH-10,

New York 10032, USA. Tel.: þ1 212 305 9562; fax: þ1 212 305 3213.E-mail addresses: Helena.Gylling@hus.fi (H. Gylling), j.plat@maastrichtuniversity.nl (J. Plat), Stephen.Turley@UTSouthwestern.edu (S. Turley), hng1@columbia.edu

(H.N. Ginsberg), lasse.ellegard@nutrition.gu.se (L. Ellegård), wendy.jessup@gmail.com (W. Jessup), Peter.Jones@umanitoba.ca (P.J. Jones), Dieter.Luetjohann@ukb.uni-bonn.de (D. Lütjohann), Winfried.Maerz@synlab.com (W. Maerz), luis.masana@urv.cat (L. Masana), guenther.silbernagel@yahoo.com (G. Silbernagel), Bart.Staels@pasteur-lille.fr (B. Staels), Jan.Boren@wlab.gu.se (J. Borén), alberico.catapano@unimi.it (A.L. Catapano), guy.debacker@ugent.be (G. De Backer), john.deanfield@gmail.com (J. Deanfield), olivierdescamps@hotmail.com (O.S. Descamps), Petri.Kovanen@wri.fi (P.T. Kovanen), riccardi@unina.it (G. Riccardi), lalet@hacettepe.edu.tr(L. Tokgözoglu), john.chapman@upmc.fr (M.J. Chapman).

1 These authors contributed equally.2 Affiliations are listed here and in Author contribution.

0021-9150 � 2013 The Authors. Published by Elsevier Ltd.http://dx.doi.org/10.1016/j.atherosclerosis.2013.11.043

Open access under CC BY-NC-ND license.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360 347

a r t i c l e i n f o

Article history:Received 11 November 2013Accepted 11 November 2013Available online 23 November 2013

Keywords:HypercholesterolaemiaPhytosterolsIntestinal cholesterol absorptionCardiovascular riskSafetyFunctional foods with added plant sterolsand plant stanols

a b s t r a c t

Objective: This EAS Consensus Panel critically appraised evidence relevant to the benefit to risk rela-tionship of functional foods with added plant sterols and/or plant stanols, as components of a healthylifestyle, to reduce plasma low-density lipoprotein-cholesterol (LDL-C) levels, and thereby lower car-diovascular risk.Methods and results: Plant sterols/stanols (when taken at 2 g/day) cause significant inhibition ofcholesterol absorption and lower LDL-C levels by between 8 and 10%. The relative proportions ofcholesterol versus sterol/stanol levels are similar in both plasma and tissue, with levels of sterols/stanolsbeing 500-/10,000-fold lower than those of cholesterol, suggesting they are handled similarly tocholesterol in most cells. Despite possible atherogenicity of marked elevations in circulating levels ofplant sterols/stanols, protective effects have been observed in some animal models of atherosclerosis.Higher plasma levels of plant sterols/stanols associated with intakes of 2 g/day in man have not beenlinked to adverse effects on health in long-term human studies. Importantly, at this dose, plant sterol/stanol-mediated LDL-C lowering is additive to that of statins in dyslipidaemic subjects, equivalent todoubling the dose of statin. The reported 6e9% lowering of plasma triglyceride by 2 g/day in hyper-triglyceridaemic patients warrants further evaluation.Conclusion: Based on LDL-C lowering and the absence of adverse signals, this EAS Consensus Panelconcludes that functional foods with plant sterols/stanols may be considered 1) in individuals with highcholesterol levels at intermediate or low global cardiovascular risk who do not qualify for pharmaco-therapy, 2) as an adjunct to pharmacologic therapy in high and very high risk patients who fail to achieveLDL-C targets on statins or are statin- intolerant, 3) and in adults and children (>6 years) with familialhypercholesterolaemia, in line with current guidance. However, it must be acknowledged that there areno randomised, controlled clinical trial data with hard end-points to establish clinical benefit from theuse of plant sterols or plant stanols.

� 2013 The Authors. Published by Elsevier Ltd. Open access under CC BY-NC-ND license.

Contents

1. Introduction and rationale . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3482. Biology and mode of action of plant sterols/stanols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 348

2.1. Origins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3482.2. Transport and circulating levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3492.3. Tissue levels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3502.4. Markers of intestinal cholesterol absorption and synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3502.5. Intestinal handling of cholesterol and non-cholesterol sterols: mechanism by which plant sterols/stanols added to the diet inhibit

the absorption of cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3503. Lipid modifying effects of plant sterols/stanols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .351

3.1. LDL cholesterol lowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3513.2. Additional effects on the plasma lipid profile . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

4. Effects on atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3524.1. Studies in animal and cell-based models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

4.1.1. Animal models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3524.1.2. Cell-based models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 352

4.2. Studies in humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3534.2.1. Relationship of plant sterol/stanol consumption and vascular health . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3534.2.2. Relationship of circulating plant sterols/stanols and CV risk: Cohort studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 353

5. Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3546. Optimising the use of plant sterols/stanols in lipid lowering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 354

6.1. Combination therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3546.1.1. Statins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3546.1.2. Ezetimibe . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3546.1.3. Other combination lipid therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

6.2. Postprandial lipaemia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3557. EAS consensus panel recommendations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 355

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 356Author contribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .357

European Atherosclerosis Society (EAS) Consensus Panel on Phytosterols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .357Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .357Supplementary material . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .357References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360348

1. Introduction and rationale humans, inhibition of cholesterol absorption by ezetimibe results inlowering of plasma LDL-C levels because of increased fractional

Fig. 1. Scheme illustrating the potential impact of cholesterol absorption from thesmall intestine on plasma levels of chylomicron remnants and LDL, with detrimentaleffects on the vascular wall. Cholesterol (Chol, C) entering the intestinal lumen pri-marily from the bile and the diet is absorbed to varying degrees and packaged inchylomicrons (CM-C) for transport via the lymph into the circulation. Therein, hy-drolysis of much of the triacylglycerol present in the nascent CM results in the for-mation of cholesterol-rich remnant particles (CMr-C) that are ordinarily rapidly clearedfrom the circulation by the liver. Delivery of excess intestinal cholesterol to the livercan result in suppression of low density lipoprotein-receptor (LDL-R) activity andendogenous cholesterol synthesis, or acceleration of hepatic very low densitylipoprotein-cholesterol (VLDL-C) secretion, or both. Such events will potentially raiseplasma LDL-cholesterol (LDL-C) concentration. If hepatic clearance of CMr is delayed,then these particles may contribute directly to plaque formation. Together, thesevarious pathways illustrate how agents that limit cholesterol absorption may elicitfavourable changes in atherogenic plasma lipoproteins that culminate in attenuatingplaque formation.

The on-going pandemic of obesity, metabolic syndrome anddiabetes is directly associated with an ever-increasing worldwideincidence of premature atherosclerosis and cardiovascular disease(CVD). Indeed, the European Union budget for CVD is currentlyestimated at V196 billion a year, with about one-half of thisattributed to direct healthcare costs [1]. However, limiting theexpenditure for CVD to the healthcare systems of the EuropeanUnion grossly underestimates its true cost. Most public health ex-penses are linked to treatment, a notion which strongly argues forurgent investment in CVD prevention to improve health in Euro-pean populations and to stem the socioeconomic consequences.

Recent studies of the relationship of lifetime risk of CVD to riskfactor burden clearly indicate that individuals with an optimal riskfactor profile, (including well-controlled blood pressure andcholesterol, non-smoking and non-diabetic), display substantiallylower risk of CV events over their lifetime than those with two ormore of these major risk factors [2]. Ranking of nine CV risk factorsin the INTERHEART cross-sectional study in 52 countries revealedthat dyslipidaemia alone accounted for most of the population-attributable risk for myocardial infarction; here, dyslipidaemiawas defined as an excess of cholesterol transported in atherogenicapolipoprotein (apo)B-containing lipoproteins (among which low-density lipoproteins [LDL] predominate), relative to that in non-atherogenic apoA-I-containing, high-density lipoproteins (HDL)[3].

Robust data attest to the causal role of LDL-cholesterol (LDL-C)in coronary atherosclerosis. Reductions in LDL-C levels achieved bytreatment with diet, statins or bile acid sequestrants, or by ilealbypass surgery, in prospective clinical trials of 3 or more yearsduration, have been demonstrated in meta-regression analyses tosignificantly reduce CV morbidity and mortality [4,5].

As atherosclerosis is a chronic, progressive disease typicallyinitiated during the first three decades of life, it follows thatlowering LDL-C early may substantially delay or even prevent theonset of atherosclerosis, particularly in the coronary circulation.Indeed, evidence that prolonged exposure to low plasma LDL-Clevels is associated with markedly greater reduction in CV riskcompared with current strategies aimed at lowering LDL-C inmiddle age, has been provided by a meta-analysis of Mendelianrandomisation studies, involving polymorphisms in six distinctgenes of cholesterol metabolism [6].

These findings prompt a key question: How can LDL-C bemaintained at low levels throughout life without imposing addi-tional burden on the healthcare system? Clearly, lifestyle, whichencompasses dietary habits, must be seriously considered, partic-ularly as extensive nutritional and behavioural changes may lowerLDL-C levels by up to 20% [7].

The liver is the principal regulator of circulating LDL-C levels.Not only is it the site of formation of very low-density lipoproteins(VLDL), the precursors of most LDL particles in the circulation, but itis also the site of most receptor-mediated clearance of LDL [8]. Boththe liver and intestine are central to body cholesterol homeostasis.Indeed, after lipolysis-mediated removal of dietary triglyceridesfrom chylomicrons, the liver rapidly clears circulating chylomicronremnants, which carry cholesterol that was absorbed in the smallintestine [9]. The resultant increase in hepatic cholesterol stimu-lates VLDL secretion and hence LDL formation, and down-regulateshepatic LDL receptor activity. Such events potentially lead to ele-vations in plasma LDL-C levels. Both chylomicron remants and VLDLremnants, in addition to LDL, can deliver cholesterol to the arterywall, initiating or exacerbating atherosclerosis. When cholesterolabsorption in the small intestinewas inhibited by ezetimibe in apoEdeficient mice, atherosclerosis was dramatically reduced [10]. In

removal of LDL from the circulation, consistent with less dietarycholesterol arriving at the liver via chylomicron remnants withsubsequent upregulation of LDL receptors [11,12]. Inhibition ofcholesterol absorption by ezetimibe was also associated withreduced chylomicron secretion into plasma as determined byreduced production of apoB48 (Fig. 1) [12].

Thus it follows that the cholesterol absorption pathway repre-sents an attractive target in themanagement of dyslipidaemia, witha specific focus on both reducing the cholesterol content of chylo-micron and VLDL remnants and lowering LDL-C levels. Thispathway presents clinical opportunities for dietary supplementa-tion with agents that attenuate intestinal cholesterol absorption,among which plant sterols and plant stanols are prominent.

The European Atherosclerosis Society (EAS) convened an inter-national Consensus Panel of basic scientists and clinical in-vestigators with expertise in cholesterol metabolism, plant steroland plant stanol biology, and CVD. Our goals were (i) to criticallyappraise state-of-the-art knowledge pertaining to the potential ofplant sterols and plant stanols (hereafter referred to as plant ste-rols/stanols) for lowering LDL-C, with a view to preventing pre-mature atherosclerosis and CVD, and (ii) to proposerecommendations for optimal integration of foods with addedplant sterols/stanols, as part of a healthy lifestyle, for attenuation ofCV risk. These recommendations can provide guidance and supportfor clinicians and health professionals in the prevention of CVDacross the spectrum of CV risk.

2. Biology and mode of action of plant sterols/stanols

2.1. Origins

Plant sterols/stanols are bioactive components with similarfunctions as that of cholesterol in mammals. Plant sterols are ste-roid alkaloids which differ from cholesterol in the structure of their

Table 1Plant sterol and plant stanol contents in different foods. Data are given as mg/100 g(dry weight, either range or mean value).

Food item Plant sterols Plant stanols

Vegetable oilsCorn oil 686e952 23e33Rapeseed oil (canola oil) 250e767 2e12Soybean oil 221e328 7Sunflower oil 263e376 4Olive oil 144e193 0.3e4Palm oil 60e78 Traces

CerealsCorn 66e178 e

Rye 71e113 12e22Wheat 45e83 17Barley 80 2Millet 77 e

Rice 72 3Oats 35e61 1

NutsPeanuts 320 e

Almond 143 e

VegetablesBroccoli 39 2Cauliflower 18e40 TracesCarrot 12e16 TracesLettuce 9e17 0.5Potato 7 0.6Tomato 7 1

Fruits and berriesAvocado 75 0.5Passion fruit 44 Not detectedRaspberry 27 0.2Orange 24 Not detectedApple 12e18 0.8Banana 12e16 Not detected

e, Not reported.Source: Adapted from Piironen V & Lampi AM (2004) [15].

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360 349

side chain, while plant stanols are 5a-saturated derivatives of plantsterols (Fig. 2).

Themain food sources of plant sterols are vegetable oils, spreadsand margarines, breads, cereals, and vegetables (Table 1); thesecontribute 50e80% of the daily plant sterol intake, with fruitsadding a further 12% [13e15]. In the typical Western diet, the meandaily intake of plant sterols is about 300 mg [13,14], but can be ashigh as 600 mg in vegetarians [16]. The most abundant are sitos-terol and campesterol, which contribute 60% and 20%, respectively,of plant sterol intake [13,14]. By comparison, the amounts of plantstanols in the diet are much lower, with only about 17e24 mg perday (predominantly sitostanol and campestanol) [14]. Cereals,especially wheat and rye, are the richest source of plant stanols.

2.2. Transport and circulating levels

These dietary components undergo low fractional absorption inthe intestine, of the order of 0.5e2% for plant sterols and 0.04e0.2%for plant stanols [17]. As a result of low absorption and efficientexcretion into bile after uptake by the liver, circulating levels arelow, varying from 7 to 24 mmol/L (0.3e1.0 mg/dL) for plant sterols,and from 0.05 to 0.3 mmol/L (0.002e0.012 mg/dL) for plant stanols[18]; these levels are of the order of 500-fold and 10,000-fold lower,respectively, than those of cholesterol.

Long-term consumption of foods with added plant sterols(mean intake � standard deviation [SD] 1.1 � 0.6 g/day) increasestheir circulating levels (from 19 to 30 mmol/L [0.8e1.2 mg/dL] withplant sterol consumption), which overlap those within the normalrange [19]. For foods with added plant stanols (mean intake0.6 � 0.4 g/day), increases in circulating levels of plant stanols(from 0.3 to 0.7 mmol/L [0.012e0.028 mg/dL] with plant stanolconsumption), and a decrease in circulating plant sterols (from 16to 23%), were observed [19]. The quantitative distribution of plantsterols/stanols across the major lipoprotein classes is similar to thatof cholesterol, and thus they circulate primarily in LDL particles(65e70%).

Fig. 2. Chemical structure of plant stanols and plant sterols.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360350

The ATP-binding cassette co-transporters G5 and G8 (ABCG5/ABCG8) play a crucial role in controlling the absorption of plantsterols/stanols by secreting absorbed molecules back into the in-testinal lumen [20,21]. In phytosterolaemia, severe loss of functionmutations in genes coding for the ABCG5/ABCG8 transportersresult in dramatic elevation in plasma plant sterol/stanol levels,which are more than 50-fold higher than those in normal in-dividuals after consumption of plant sterols, and many of theseindividuals develop premature atherosclerosis (Box 1) [20e22].Further consideration of this rare genetic disease is, however,beyond the scope of the present paper, which is focussed on plantsterol/stanol consumption as part of a healthy diet for prevention ofCVD.

2.3. Tissue levels

There are limited data available for tissue levels in subjectsreceiving foods with added plant sterols/stanols. In healthy humansubjects, plant sterols/stanols are taken up into tissues in similarproportions relative to cholesterol, and the ratio of cholesterol toplant sterols/stanols, which displays a wide range (0.001e0.01 � 10�4), is similar to or less than that in plasma, consistentwith the absence of preferential accumulation or retention in tis-sues (see Supplementary Table 1). Similar findings have been re-ported for cerebrospinal fluid and brain tissue in healthy controlsubjects, and also in phytosterolaemia [23,24]. In healthy volun-teers, data on tissue plant sterol/stanol levels are available for thecarotid artery wall and for non-stenotic and stenotic aortic valvecusps [25e28]. In these tissues, cholesterol content is 20 mg/mg oftissue, while total plant sterol and plant stanol concentrations arew0.04 mg/mg andw0.001 mg/mg of tissue, respectively. Plant sterolconcentrations vary widely between tissues (see SupplementaryTable 1) [25e30].

Box 1

Phytosterolaemia

� Phytosterolaemia (previously referred to as sitoster-

olaemia) is due to rare, loss of function mutations in

genes coding for the ATP-binding cassette transporters

G5 and G8 (ABCG5/ABCG8), and is characterised by very

high serum levels of plant sterols (up to 1.3 mmol/L or

50 mg/dL) and plant stanols (0.2 mmol/L or 8 mg/dL) [18].

� Major clinical manifestations may include premature

atherosclerosis although this complication is variable;

there are a number of patients with phytosterolaemia who

do not have evidence of atherosclerosis (D. Lutjohann,personal communication; E. Bruckert, privileged commu-nication). The presence of premature atherosclerosis ap-

pears to depend on whether there is co-existing severe

hypercholesterolaemia. Other complications of phytos-

terolaemia include episodes of haemolysis, and xantho-

mas. Moreover, it is noteworthy that recent findings in 4

adult phytosterolaemic patients have not detected any

significant degree of atherosclerosis (E. Bruckert, privi-leged communication).

� Plant sterols have been shown to accumulate in athero-

sclerotic lesions of phytosterolaemic subjects in the same

ratio as present in serum. However, the phytosterol/

cholesterol ratio is higher in phytosterolaemia than in

normal subjects in plasma and in tissues.

� The potential relevance of markedly elevated amounts of

plasma and tissue plant sterols and plant stanols to tissue

deposition and the atherosclerotic process remains

indeterminate.

During consumption of plant sterol-enriched foods, there was a5-fold elevation in campesterol in stenotic aortic valve cusps [28].However, in another study, the concentration of plant stanols in thearterial wall was not modified during consumption of foodsenriched with plant stanols [27]. Clearly, there is a need for large,long-term clinical studies to exclude the possibility that con-sumption of dietary plant sterols/stanols might result in accumu-lation in arterial tissues, and evaluation of these agents asmodulators of plaque formation or regression is equally of interest.Such studies present multiple methodological challenges, not leastof which is access to plaque tissue for analysis of sterol composi-tion, and for determination of the expression profile of key genes ofcholesterol and plant sterol metabolism.

2.4. Markers of intestinal cholesterol absorption and synthesis

Serum cholesterol precursors and plant sterols, especially whenexpressed as ratios relative to cholesterol, can constitute markers ofthe rates of synthesis and absorption of cholesterol in non-dyslipidaemic subjects, as well as in several clinical conditionsincluding primary and familial hypercholesterolaemia (FH), obesityand type 2 diabetes, and during some interventions including plantstanol consumption [31,32]. Overall, such markers have good val-idity, but there are exceptions (see Supplementary Appendix) [31e33], and full validation in multiple conditions is essential. More-over, there are a number of methodological issues relating tostandardisation of these markers. An on-going survey is investi-gating the degree of variability in sterol/stanol analytical dataacross different laboratories (see Supplementary Appendix).

2.5. Intestinal handling of cholesterol and non-cholesterol sterols:mechanism by which plant sterols/stanols added to the diet inhibitthe absorption of cholesterol

The handling of sterols and stanols entering the intestinal lumenfrom the diet and bile is essentially a triphasic process (Fig. 3) [34e37]. The first phase is largely physicochemical in nature, occursintraluminally, and culminates in the incorporation of cholesteroland other sterols and stanols into mixed micelles that serve asvehicles to carry these poorly water-soluble substances up to thesurface of the brush border membrane on the enterocyte. Thissolubilisation step is essential for the subsequent entry of any typeof sterol into the absorptive cell, from where it may potentiallyreach the circulation.

The second major phase is the uptake of cholesterol and othersterols or stanols into the enterocyte, a process that is facilitated bya plasma membrane-localised, general sterol transporter protein,Niemann-Pick C1-Like1 (NPC1L1). Within the absorptive cells,sterols undergo different fates, depending mainly on their chemicalstructure. Collectively, these events within the enterocyte broadlyconstitute the third major phase of intestinal handling. Thebulk of the cholesterol is esterified by acyl CoA: cholesterolacyltransferase-2 (ACAT2) and incorporated into nascent chylomi-crons, which initially enter the lymph before joining the circulationvia the thoracic duct [38,39]. The absolute content of plant sterols/stanols in chylomicrons is markedly lower than that of cholesterol,with up to 50% in esterified form [39]. Importantly, the bulk of plantsterols/stanols is pumped back into the gut lumen via the ABCG5/ABCG8 transporter, resulting in minimal entry of these plant-derived molecules into the circulation [34,40,41]. Finally, foodswith added gram quantities of plant sterols/stanols cause a signif-icant inhibition of cholesterol absorption, most likely throughdisruption of the intraluminal solubilisation step [37], althoughother possible mechanistic explanations have been proposed [42].

Fig. 3. Schematic summarising the three main steps (indicated by the numbers in red circles) involved in the intestinal handling of cholesterol and non-cholesterol sterols. In thefirst step within the intestinal lumen, sterols are incorporated into mixed micelles (1). At the brush border membrane, sterols are released from the micelles and transported intothe cell via the Niemann-Pick C1-Like1 transporter (NPC1L1) (2). Once internalised, these sterols can be handled in different ways (3). For the bulk of the internalised non-cholesterol sterols, efflux back into the lumen occurs via ABCG5/ABCG8 (ATP binding cassette transporters G5 and G8). In contrast, a significant proportion of the internalisedcholesterol undergoes esterification via ACAT2 (acyl CoA-cholesterol acyltransferase-2). The esterified cholesterol, along with much smaller quantities of esterified non-cholesterolsterols, are incorporated into nascent chylomicrons which enter the lymphatic system and ultimately the circulation. Dietary plant sterol or stanol supplementation is believed toinhibit the absorption of cholesterol most likely through disruption of the intraluminal solubilisation step [37]. Abbreviations: apo apolipoprotein; C cholesterol; CE cholesterylester; FA fatty acid, MTP microsomal triglyceride transfer protein; TG triglyceride.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360 351

As discussed above, the ABCG5/G8 genes play a crucial role inregulating circulating levels of plant sterols [20,21]. In the KORAstudy, common variants of rs41360247 and rs4245791 in the ABCG8gene accounted for 8% of the variance in circulating levels of plantsterols [43]. The relationships of these and other variants in highlinkage disequilibrium with circulating plant sterol levels havebeen confirmed in the CARLA, LURIC, and YFS cohorts [43,44].

Box 2

Types of foods with added plant sterols or plant stanols

Low-fat spread/margarine

Yoghurt drinks

Dairy-free drinks

Low- or reduced-fat milk

Soft cheese

Orange juice

Muesli

Bread

Biscuits

3. Lipid modifying effects of plant sterols/stanols

3.1. LDL cholesterol lowering

Epidemiological studies in the UK (n ¼ 22,256), Sweden(n ¼ 77,652) and China (n ¼ 3940) observed that naturally-occurring dietary plant sterol intake is inversely related to plasmatotal- and LDL-C levels [45e47]. However, in a well-controlledstudy in healthy subjects, low (126 mg plant sterols/2000 kilocal-ories) or high intake of plant sterols (449 mg plant sterols/2000kilocalories) did not affect plasma LDL-C concentrations in spite ofmodulating cholesterol metabolism [48]. Furthermore, even at thehighest levels of dietary intake, plant sterols/stanols occurringnaturally in the diet have a modest hypocholesterolaemic effect. Onthe other hand, when natural plant sterols were omitted from thediet, serum LDL-C concentrations increased [49].

In contrast to the minimal effects of variation in consumption ofnaturally-occurring plant sterols/stanols in the diet, early studiessuch as that by Farquhar et al. (1956) demonstrated that beta-sitosterol supplementation lowered both total serum cholesteroland LDL-C (as beta-lipoprotein lipid) in young men with athero-sclerotic heart disease [50]. Subsequently, Miettinen et al. (1995)demonstrated for the first time that foods (such as margarine, seeBox 2) enriched with sitostanol ester lowered both total serumcholesterol and LDL-C in mildly hypercholesterolaemic subjects[51].

Subsequent data show consistent support for the LDL-Clowering effects of foods with added plant sterols/stanols [52e

54]. In the most recent meta-analysis, consumption of foods withadded plant sterols/stanols (2 g/day) lowered LDL-C to a similarextent (8.2% and 9.3%, respectively) [54]. Regrettably, there is apaucity of data relating to the potential of higher plant sterol/stanoldoses to further lower LDL-C levels, and thus CV risk [6,54,55]. It hasbeen suggested that maximal LDL-C lowering may be greater withplant stanols (up to 16%) and plant stanol esters (17%), but thisconclusion relies on limited studies using doses of 4e9 g/day, noneof which involved direct head-to-head comparisons with plantsterols. Whether there is a position for plant sterol and/or plantstanol intakes to be raised higher than those currently recom-mended (2e3 g/day) in prevention strategies for the general pop-ulation remains open. The fact that there is consistent, robustevidence indicating that lowering of LDL-C by different mecha-nisms (statins, diet, partial ileal bypass, bile acid sequestrants) re-sults in reduction in CV risk, underlies the rationale for inclusion ofplant sterols/stanols in international clinical guidelines for themanagement of dyslipidaemia [5,56]. In this context, it must berecognised that the use of functional foods enriched with plantsterols/stanols is currently not advised for children under 6 years.There is, however, a substantial database showing consistent LDL-Clowering efficacy in children. In controlled clinical trials in children

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360352

and adolescents aged 4e15 years, consumption of foods supple-mented with plant sterols/stanols (1.5e3 g/day) resulted inconsistent LDL-C reduction (by 5e15%) in normolipaemic children(n ¼ 98), and equally in children with FH (n ¼ 224; by 9e19%) (seeSupplementary Table 2) [57e66]. Themagnitude of such reductionsis comparable to that observed in adults. On this basis, the Panelproposes that dietary supplementation with plant sterols/stanolsmay be considered in children (from the age of 6 years) with FHwho require lipid lowering treatment as an adjunct to lifestyleadvice and potential pharmacotherapy, although long term safetystudies are clearly needed.

3.2. Additional effects on the plasma lipid profile

Most studies with plant sterols/stanols were conducted in in-dividuals with isolated hypercholesterolaemia. The available datasuggest that triglyceride levels are reduced by 6e20% at intakes of1.5e2 g/day of plant sterol/stanol, with essentially no effect onHDL-C [67e69]. Pooled analyses showed a modest reduction inplasma triglycerides of 6% and 4% for recommended intakes of plantsterols (1.6e2.5 g/day) or plant stanols (2 g/day), respectively[69,70]. Indeed, evidence suggests a relationship between baselinetriglyceride levels and the magnitude of this effect, with 9%reduction when baseline triglycerides were 1.9 mmol/L (170 mg/dL), but no effect at baseline levels of 1.0 mmol/L (90mg/dL) [69]. Insubjects with metabolic syndrome and moderate hyper-triglyceridaemia, plant stanols lowered hepatic production of bothlarge (>60 nm) and medium size (35e60 nm) VLDL particles [67].Other studies documented a reduction in small, dense LDL particlesin patients with type 2 diabetes or metabolic syndrome after con-sumption of plant stanols/sterols [68,71]. Based on one study, itappears that plant sterols/stanols do not influence lipoprotein(a)levels [72].

Finally, it is of considerable interest that LDL-C reduction, sub-sequent to consumption of a plant stanol ester-enriched diet in apopulation of metabolic syndrome subjects, was without effect onplasma levels of proprotein convertase subtilisin/kexin type 9(PCSK9) (J Plat, unpublished data), a potential contrast to theelevation in PCSK9 levels induced upon statin-mediated LDL-Clowering [73].

Future studies of the potential effects of foods with added plantsterols/stanols in attenuating the atherogenicity of the postprandialperiod in well-phenotyped cohorts of subjects with metabolicsyndrome or type 2 diabetes would be of special interest. Suchinvestigations should focus on normalisation of both the qualitativeand quantitative features of atherogenic triglyceride-rich lipopro-teins and their remnants during this phase.

4. Effects on atherosclerosis

4.1. Studies in animal and cell-based models

Because plant sterols/stanols effectively reduce plasma LDL-Cconcentrations, their use could constitute a potential protectivestrategy against the initiation and progression of atherosclerosis.Most of the available supportive data relate to studies in animal-and cell-based models, however, these have inherent limitations.Thus, investigations in animal models have been of short duration,while very high (pharmacological) doses have been typicallyapplied in both animal and cell models, thereby highlighting theneed for cautious interpretation of the experimental findings.

4.1.1. Animal modelsTo date, more than 30 studies have investigated the effect

of plant sterol/stanol supplementation on experimental

atherosclerosis in various animal models (see SupplementaryTable 3 and reviewed by Kritchevsky and Chen [2005]) [74]. Ingenetically-modified mouse models of atherosclerosis, protectiveeffects were observed despite increases (up to 10-fold) in plasmaplant sterol/stanol concentrations [28,75,76]. Such effects includedreduction in arterial lipid accumulation, and inhibition of lesionformation and progression. Moreover, regression of existing lesionscorrelated with the cholesterol-lowering action of plant sterols/stanols.

4.1.2. Cell-based models4.1.2.1. Cellular metabolism of plant sterols/stanols and potentialimpact on cholesterol metabolism. The proportions of plant sterols/stanols relative to cholesterol found in plasma are maintained intissue sterols [24,26], suggesting that they are handled similarly tocholesterol inmost cells. While direct studies of cellular plant sterolmetabolism are relatively limited in scope, they support this view.For example, the rates of uptake and accumulation of sitosterol andcholesterol by macrophages are similar [77], and substantialesterification of beta-sitosterol (¼sitosterol) and other plant sterolscan be measured in many tissues and cells [26,78,79]. On the basisof limited data, efflux of sitosterol and sitostanol to HDL from hu-man macrophages appears to be more efficient than that ofcholesterol [77].

Cholesterol homeostasis is tightly controlled at the transcrip-tional level via Sterol Regulatory Element-Binding Protein-2 andliver X receptor (LXR)-dependent regulation, and also by post-translational control of the turnover of a number of key enzymes,receptors and transporters. Plant sterols appear to have little or noimpact on these processes. Their ability to activate LXR-dependentgenes is negligible or very low [80e84]. A plant sterol-enriched diethad no effect on mouse intestinal LXR target gene expression [85].In future studies, more comprehensive evaluation of the effects of‘physiological’ concentrations of plant sterols/stanols on the he-patic and intestinal gene expression profile would be of consider-able interest.

4.1.2.2. Influence of plant sterols/stanols on inflammatory pathways.In macrophages, recent studies have shown that lipid accumulationand inflammatory responses are co-ordinated through LXR-mediated transrepression of inflammatory genes by desmosterol,an intermediate in cholesterol synthesis and an endogenous LXRligand [86,87]. Interestingly, as plant sterol/stanol consumptionresults in a compensatory increase in endogenous cholesterolsynthesis [31], the corresponding increase in intracellular des-mosterol concentrations could result in plant sterol/stanol-inducedanti-inflammatory effects. Indeed, a number of in vitro studiesindicate that some plant sterols exert anti-inflammatory effects onactivated macrophages. However, these studies have significantlimitations, as they involved addition of plant sterols and/or stanolsunder conditions, which (although seldom directly measured),almost certainly significantly increase the plant sterol:cholesterolratio to values well in excess of the normal 1:500 to 1:1000 ratiothat exists in vivo. This is likely evenwhen plant sterols are added at‘physiological’ concentrations. Future studies must include directmeasurement of the cellular levels of cholesterol and plant sterolsand should aim to mimic those found in vivo.

There are conflicting findings with respect to the effects ofsitosterol and campesterol on production of proinflammatory fac-tors by macrophages [88e92]. Interestingly, sitosterol stimulatedanti-oxidant pathways and inhibited either phorbol ester- orlipopolysaccharide-stimulated prostaglandin synthesis in mousemacrophages [93e96]. The potential relevance of these findings tothe atherosclerotic process is unclear at present, but warrantsfurther research. In addition to macrophages, substantial evidence

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360 353

attests to the implication of T-lymphocytes in the immuno-inflammatory dimension of atherosclerosis [97]. Interestingly,both plant sterols and plant stanols exert immune-modulatingproperties, to the extent that the evoked T-helper cell (Th) 1response, as shown by enhanced production of the Th1 cytokines,interferon-gamma and interleukin-2, and activation of toll receptor2, is an essential feature of this response [92].

Thus, in summary and based on the evidence discussed, wecannot exclude the possibility that accumulation of plant sterols/stanolsmight occur in vascular cells as a consequence of an increasein their circulating concentrations (see Box 3).

4.2. Studies in humans

4.2.1. Relationship of plant sterol/stanol consumption and vascularhealth

Human studies evaluating the effect of dietary plant sterols/stanols on measures of arterial structure or function using carotidintima media thickness, brachial artery size, flow-mediated dila-tation (FMD), and arterial stiffness have not shown any major,consistent effects on vascular or endothelial function during shortandmid-term plant sterol or plant stanol intake [64,66,98e103]. Allstudies were small and mostly of short duration, and importantly,several studies were in subjects with low CV risk exhibiting normalvascular function at baseline. Despite significant reductions in LDL-C levels, no consistent changes in measures of inflammation,oxidative stress or endothelial functionwere observed. In a study ofapparently healthy non-smokers, daily use of plant stanol marga-rine for �2 years did not improve carotid artery compliance [103],and in a shorter (3 months) study, failed to impact FMD [98].Similarly, in two studies in pre-pubertal children with FH, dietaryplant sterol/stanol consumption did not improve endothelialfunction despite significant LDL-C lowering [64,66].

Recent research has focused on the characteristics of themicrocirculation as markers of early arterial disease [104]. In onetrial [105], intake of foods enriched with plant sterols/stanols(2.5 g/day) for 85 weeks by statin-treated subjects showed an as-sociation between the increase in serum campesterol concentra-tions and changes in both retinal arteriolar and venular diameters.Retinal venular diameters increased (by 2.3� 3.1 mm) and arteriolardiameter decreased (by 0.7 � 4.7 mm) in the group receiving addedplant sterols, although these effects did not reach significance.However, the change in cholesterol-standardised campesterolconcentrations correlated positively with the change in venulardiameter independent of changes in LDL-C concentration. Otherstudies with endpoints including coagulation [72], platelet

Box 3

Implications from studies in animal- and cell-based models of

the impact of plant sterols and plant stanols on atherosclerosis

� The limited available data indicate that most cells handle

(uptake, esterification, export) plant sterols/stanols in a

similar manner as cholesterol.

� In vivo, plant sterols represent w0.1% of total cellular

sterol; however, most in vitro studies have involved the

use of much higher levels of plant sterols.

� Evidence suggests that plant sterols do not affect cellular

cholesterol homeostasis, although further studies are

needed to confirm this.

� Some studies indicate mild anti-inflammatory effects of

plant sterols/stanols, but confirmation is required at

‘physiological’ levels of plant sterols.

� Plant sterols are not toxic at ‘physiological’ concentrations.

aggregation measures [106], oxidant stress [107], inflammation[108] or other biomarkers did not demonstrate convincing benefit.It is possible, however, that the reduction of LDL-C by 10%, astypically mediated by intake of foods with added plant sterols/stanols, may not attain the threshold required for impact on theseparameters. Adequately powered, controlled studies of the effectsof plant sterols/stanols on morphological, functional andbiochemical surrogatemarkers of atherosclerosis are needed. Basedon present evidence, there is no indication that dietary supple-mentation with plant sterols/stanols are associated with eitherbenefit or harm to vascular function.

4.2.2. Relationship of circulating plant sterols/stanols and CV risk:Cohort studies

Several observational studies have investigated the associationbetween circulating plant sterols and atherosclerosis in the generalpopulation. Some early reports found moderately elevated plantsterol levels to be positively associated with vascular disease[109,110], although others suggested an inverse or lack of rela-tionship between circulating plant sterols and CV risk [111e113]. Arecent meta-analysis including 17 studies (n ¼ 11,182) [114],showed no significant associations of circulating campesterol andsitosterol with vascular disease over a range of circulating plantsterol concentrations (average concentrations for first and thirdtertiles, 0.17 and 0.47 mg/dL [4 and 12 mmol/L] for campesterol, and0.13 and 0.38 mg/dL [3 and 10 mmol/L] for sitosterol). Two studiesshowed inverse relationships between plant sterols and CV risk,one study did not find any association, and a further study reporteda positive association between plant sterols and CV risk [115e118].

Since these retrospective/case-control and prospective/cohortstudies do not provide the highest level of evidence in definingcausality, and as placebo-controlled trials with these endpoints arelacking, Mendelian randomisation studies may be informative.Moderate elevations of circulating plant sterols, which are associ-ated with plant sterol-raising variants in the ABCG8 gene, showed apositive association with prevalent coronary artery disease[43,119e122]. Such an increase in risk can, however, be entirelyexplained by the association of ABCG8 variants with intestinalcholesterol absorption, as reflected by circulating cholestanollevels, and with LDL-C plasma concentration, rather than circu-lating levels of plant sterols [44,123e126]. The ABO gene has alsobeen associated with elevated circulating sterol levels and CV risk[43]. However, the ABO locus exhibits even greater pleiotropy thanthe ABCG5/G8 locus, as it regulates von Willebrand factor, coagu-lation factor VIII, intercellular adhesion molecule-1, P-selectin, andE-selectin levels [127e129]. In the largest report to date (n¼ 1242),circulating levels of plant sterols were lower in the group withcoronary artery disease (CAD), and concentrations of the plantstanols, campestanol and sitostanol, which are much lower thanthose of plant sterols, were not different between the groups withand without CAD [130]. Thus, the available Mendelian random-isation studies do not provide a scientific basis to discourage theuse of plant sterol- or plant stanol-containing functional foods.

A healthy diet is the cornerstone of CVD prevention. In thisrespect, it is of critical importance that indisputable evidencesupports the contention that lowering of plasma LDL-C levelsconfers clinical benefit, irrespective of mechanism, and that suchmechanisms include dietary intervention [4e7,131e133]. Indeed,robust data show that there is no qualitative difference betweenstatin- and non-statin-mediated reduction in LDL-C whencomparing their estimated effects on myocardial infarction or CHDdeath on the basis of the Bayes factor [5]. Thus, the regression linesfor all individual diet (n ¼ 5), bile acid sequestrant (n ¼ 3), surgery(n ¼ 1), and statin (n ¼ 10) trials were similar and consistent with aone-to-one relationship between LDL-C lowering and reduction in

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360354

CHD and stroke over 5 years of treatment [5]. In this context, and asdiscussed above, it is of immediate relevance that consumption of2 g/day of plant sterols/stanols, as part of a healthy diet, lowers LDL-C plasma levels by approximately 10% [52e54].

The EAS Consensus Panel on Phytosterols, however, recognisesthat there is a lack of randomised data pertaining specifically to theimpact of foods with added plant sterols/stanols on CVD preven-tion. Large-scale outcome trials of food products with added plantsterols/stanols for CVD prevention in the setting of low to inter-mediate risk are not practically feasible, given the very largenumber of subjects (>50,000) required for adequate power (seeSupplementary Appendix 2. Feasibility of an outcomes study forfood products with added plant sterols/stanols). Furthermore,the benefit of consistent, but relatively small, additional LDL-Clowering from consumption of foods with added plant sterols/stanols, as part of a healthy diet, would be difficult to demonstratedefinitively in a clinical trial of optimally treated patients at high CVrisk, even if 25e30,000 individuals were enrolled. The absence ofrandomised, controlled trial data with hard end-points to establishclinical benefit from the use of plant sterols or plant stanols must beconsidered when health professionals choose to advise their use assupplements to a healthy diet.

5. Safety

In the context of benefit-risk considerations, substantial interesthas focused on the safety aspects of plant sterols/stanols when usedas cholesterol-lowering agents. Given that the level of plant sterol/stanol intake required for cholesterol lowering ranges from 1 to 3 g/day, well above typical consumption patterns which rarely exceed400e600 mg/day, a considerable literature has amassed exploringpossible deleterious effects of prolonged plant sterol/stanol con-sumption. Indeed, for some individuals zealously consuming mul-tiple foods with added plant sterols/stanols, it is feasible thatintakes could rise considerably above the 3 g/day level consideredto represent the ceiling at which the dose-response curve forcholesterol-lowering with plant sterols begins to plateau. However,evidence from clinical studies and post-launch monitoring in-dicates that overconsumption of foods with added plant sterols/stanols is not an issue [19,134].

Specific areas of concern surrounding plant sterol/stanol con-sumption include (i) possible negative effects on fat-solublevitamin status, and (ii) and cancer risk. Overall, longer-term post-launch monitoring efforts have failed to answer the questionwhether foods with added plant sterols/stanols cause any unex-pected negative health effects [135,136]. Several longer-termfeeding trials have similarly failed to identify any negative actionof plant sterol/stanol consumption on clinical chemistry, haema-tology or clinical symptomology [52e54,137,138].

A repeated observation in some, but not all, plant sterol feedingtrials is modest suppression of plasma carotenoid concentrations(by 10%), especially for the highly lipophilic hydrocarbon caroten-oids (beta-carotene, alpha-carotene and lycopene) [139,140]. It hasbeen suggested that this may arise from suppression of intestinalabsorption [141]. Increasing consumption of fruits and vegetablesoffsets any decline in fat-soluble vitamin levels induced by plantsterol/stanol intakes in the range that lowers cholesterol [139].

Considerable controversy has occurred over the past decaderegarding a possible atherogenic role of high circulatory levels ofplant sterols/stanols. Reports are conflicting, although the mostrecent work seems to indicate that plasma levels of plant sterolsassociated with recommended intakes (2 g/day) do not pose ahealth risk. Mendelian randomisation studies have claimed to showthat increased circulatory levels of plant sterols increase CVD risk,but since the ABCG5/G8 polymorphisms examined have pleiotropic

effects, the results of these studies can readily be explained by anincreased cholesterol absorption rate (see above).

Lastly, considerable evidence from animal and cell studies sug-gests, if anything, a protective role of sitosterol against certaincancers [142e145]. Thus, there is no increase in cancer risk withrecommended daily intakes of functional foods containing 2e3 g/day of plant sterols/stanols. In fact, surveillance data associatereduced risk of certain cancers with plant sterol/stanol intakes[146,147]. Possible mechanisms based on animal and cell worksuggest direct actions on apoptosis or indirect actions through theintracellular cholesterol-lowering ability of these agents [145].

Overall, evidence from longer-term monitoring trials, as well asexperimental models, indicates that plant sterols/stanols present afavourable safety profile, thereby supporting their use in choles-terol lowering, either alone or adjunctive to pharmacotherapy.Based on the epidemiology, the genetics, and the wealth of currentclinical trial evidence demonstrating LDL-C lowering with plantsterols/stanols and lack of significant safety concerns, the use ofplant sterols/stanols in treating hypercholesterolaemia can beencouraged [56]. The question remains how best to optimise theiruse in lipid management.

6. Optimising the use of plant sterols/stanols in lipid lowering

6.1. Combination therapy

In addition to a role in primary prevention in the general pop-ulation, foods with added plant sterols/stanols may provide addi-tional LDL-C lowering in dyslipidaemic patients at high CV risktreated with lipid-lowering drugs. Thus, it is important to definethe lipid-modifying effects of dietary plant sterols/stanols (2e3 g/day) in combination with pharmacotherapies so as to optimisetheir clinical use.

6.1.1. StatinsStatins are inhibitors of the rate-limiting enzyme of cholesterol

biosynthesis, HMG-CoA reductase. As such, their action directlydecreases intracellular levels of cholesterol and its precursors, en-hances the catabolism of apoB-containing lipoproteins (mainlyLDL) via upregulation of hepatic LDL receptors, and reduces de novohepatic (and potentially intestinal) lipoprotein production. Sinceplant sterols/stanols act via a distinct mechanism, i.e. by loweringbioavailability of intestinal cholesterol for entry into the circulation,it can be speculated that plant sterols/stanols may exert an additiveeffect when combinedwith a statin. In clinical studies, dietary plantsterols/stanols induce an incremental decrease in LDL-C levels of10e15% when added on top of statin therapy, which is superior tothat (6%) obtained by doubling the statin dose [148e151]. In vivostudies of LDL-apoB kinetics in patients with type 2 diabetes mel-litus indicated that the additive effects on LDL-C reduction whenstanols were added to statins resulted from decreased productionof LDL [152]. Thus, the generally accepted efficacy of plant sterol/stanol consumption (2e3 g/day) is maintained on top of statintherapy.

6.1.2. EzetimibeAs the lipid-lowering mechanism of ezetimibe is mediated by

inhibition of intestinal cholesterol absorption, ezetimibe could beconsidered a competitor of dietary plant sterols/stanols at themolecular level. Importantly, however, the targets differ; ezetimibeblocks the NPC1L1 transporter, while plant sterols/stanols displacecholesterol from intestinal micelles. Moreover, as NPC1L1 is also theentry gate for dietary plant sterols/stanols into the body, ezetimibe-mediated inhibition of this mechanism should both enhance theireffects in the lumen of the intestine, and also reduce their plasma

Box 4

Consensus panel recommendations

� Daily consumption of foods with added plant sterols and/

or plant stanols in amounts of up to 2 g/day is equally

effective in lowering plasma atherogenic LDL-C levels by

up to 10%, and thus may be considered as an adjunct to

lifestyle in subjects at all levels of CV risk. At higher daily

intakes (9 g/day), the effects of plant stanols appear more

pronounced, but additional studies are needed to confirm

these results and examine safety at higher doses.

� Plant sterols and plant stanols can be efficaciously com-

binedwith statins. Very limited data suggest plant sterols/

stanolsmay also lower LDL-C levels in combinationwith a

fibrate or ezetimibe. In this way, the potential for attain-

ment of LDL-C goals as a function of overall CV risk can be

enhanced.

� Enhanced consumption of plant sterols and plant stanols

may be considered as an adjunct to lifestyle and dietary

approaches for modestly reducing elevated plasma tri-

glyceride levels, especially when levels are elevated

before treatment. This needs further study in appropriate

populations with elevated triglycerides.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360 355

levels. However, clinical data are limited, and in the largest study todate, there was a significant incremental reduction in intestinalcholesterol absorption during administration of ezetimibe plusplant sterols (2 g/day) associated with a significant 8% reduction inLDL-C compared to ezetimibe alone [153].

6.1.3. Other combination lipid therapyThe relevance of other combination therapies merits consider-

ation, especially from the perspective of comprehensive lipid con-trol in cardiometabolic disease. Clinical studies show a trend for anadditive effect on LDL-C levels when foods enriched with plantsterols/stanols are consumed with a fibrate [154,155]. Given thatN � 3 fatty acids have a small effect on cholesterol metabolism andmainly influence triglyceride levels, consumption of plant sterols/stanols and N � 3 fatty acids may exert a complementary beneficialeffect on the lipid profile. Indeed, both are recommended as

Fig. 4. Addition of functional foods with plant sterols/stanols, as a component of lifestylemediate or low global cardiovascular risk who do not qualify for pharmacotherapy (as indDyslipidaemia [56]. Equally, foods with added plant sterols/stanols may be considered in theindicated by blue shading). Adapted from Reiner et al. (2011) [56].

components of a healthy diet for prevention of CVD [56,156].Currently there are insufficient data regarding the lipid-loweringefficacy of a combination of bile acid sequestrants and dietaryplant sterols/stanols. However, as bile acid sequestrants interactwith lipophilic substances, it is likely that such a combination willinterfere with intestinal sterol/stanol absorption. Additionally, di-etary plant sterols, but not plant stanols, suppressed bile acidsynthesis, probably altering bile acid sequestrant-mediatedcholesterol-lowering efficacy [157].

In conclusion, the 10% reduction in serum LDL-C concentrationstypical of consumption of foods with added plant sterols/stanols atdoses of approximately 2 g/day, persists on top of the effect ofstatins. Limited data suggest an additive effect of plant sterols/stanols with ezetimibe and fibrates.

6.2. Postprandial lipaemia

Since foods with added plant sterols/stanols reduce cholesterolabsorption, they might also reduce the production of intestinally-derived chylomicrons and chylomicron remnants. The few studiesthat have been conducted, using either a single standardised mealor day-long measures of plasma triglycerides, have not shown anyeffect of either acute or chronic plant sterol/stanol intake on post-prandial triglycerides [150,158,159]. Where measured, levels ofpostprandial plant sterols/stanols were variable [158,159]. Inter-estingly, consumption of plant sterols esterified with fish oil for onemonth resulted in lower postprandial triglyceride levels than fishoils alone [160]. Overall, the limited data examining the potentiallowering of postprandial triglyceride excursions do not show sig-nificant effects, which might relate to low levels of baseline tri-glycerides in subjects in these studies.

7. EAS consensus panel recommendations

Currently, foods with added plant sterols/stanols may beconsidered in individuals with high cholesterol levels but equally inthosewith intermediate or lowglobal CV risk who do not qualify forpharmacotherapy [56]. On the basis of critical appraisal of the ev-idence base above, this EAS Consensus Panel therefore considersthat there is a place for these products, in conjunction with other

intervention, may have potential value in individuals with high LDL-C levels at inter-icated by pink shading), in line with the joint ESC/EAS Guidelines for Management ofcontext of lifestyle intervention in subjects at high or very high cardiovascular risk (as

Table 2Comparison of the cost of foods with and without added plant sterols. Data based ona strategic analysis of the European market (Frost & Sullivan Research Service,London, UK, 2005) [163].

Product type Cost per kg (UK sterling) Incrementalcost ratioa

Foods with addedplant sterols

Foods without addedplant sterols

Spreads 7.46e7.98 1.80 4.14e4.43Health drinks 4.95e8.10 2.20 2.25e3.68Yoghurt 2.80e4.00 2.10 1.33e1.90

a Ratio of cost of foods with added plant sterols to cost of foods without plantsterols.

Box 6

Health economic evaluation of consumption of foods with added

plant sterols and/or plant stanols

Nutrition economics follows a 4-step process in order to

compute the health and economic impact of penetration of

the market place of foods with added plant sterols/stanols.

To adopt the 4-step cost-of-illness approach:

� Determine a success rate for adoption of foods with

added plant sterols/stanols across the target population

� Evaluate the extent of LDL-C reduction due to consump-

tion of foods with added plant sterols/stanols

� Assess the decrease in coronary heart disease (CHD)

prevalence due to the estimated reduction in plasma LDL-

C levels

� Estimate the healthcare savings resulting from the

reduction in CHD prevalence

This multistep approach can be applied with a range of

inputted values for the 4 steps above, ranging from opti-

mistic to pessimistic.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360356

lifestyle interventions, in patients receiving lipid-lowering therapywith statins or other agents who do not achieve LDL-C targets, or inthose with statin intolerance (see Box 4 and Fig. 4). Finally, giventhe increasing importance of early preventive strategies in hyper-cholesterolaemia [6], the potential for inclusion of plant sterol- orplant stanol -enriched foods in the diet of adults and children (>6years) with FH, as an adjunct to lifestyle and pharmacotherapy, maybe considered. We base these recommendations on the provenability of plant sterols/stanols to lower plasma LDL-C levels in theabsence or presence of concomitant statin therapy, and equally onevidence that they reduce plaque size in atherosclerosis-proneanimal models. We understand that, in the absence of CVDoutcome data from randomised clinical trials, the evidence for useof plant sterols/stanols is incomplete.

Healthcare professionals should take into account the level ofoverall CV risk of patients, and their preferences. In addition, the

Box 5

Unresolved questions

� Does inter-individual variability occur in the cholesterol-

lowering efficacy of plant sterols/stanols, i.e. is it

possible to identify hyper- versus hypo-responders? If so,

is it also possible to overcome lack of or poor respon-

siveness with higher plant sterol/stanol intake?

� Do plant sterols/stanols affect lipoproteins beyond LDL-C,

i.e. atherogenic triglyceride-rich lipoproteins and their

remnants, LDL and HDL subfractions, and lipoprotein(a)?

� Do plant sterols/stanols reduce the potential atheroge-

nicity of postprandial lipid and lipoproteins, especially in

subjects with cardiometabolic disease such as type 2

diabetes?

� How do plant sterols/stanols affect lipoprotein meta-

bolism in specific populations, for instance in patients

with the metabolic syndrome or type 2 diabetes mellitus

who exhibit an atherogenic lipoprotein phenotype?

� How do plant sterols affect cellular lipid homeostasis?

� How do plant sterols affect the function of cells involved

in the development of atherosclerosis, such as endothe-

lial cells, monocytes and macrophages under resting and

‘activated’ conditions in vitro, ex vivo and in vivo?� Do plant sterols/stanols have significant effects on

biochemical surrogate markers of atherosclerosis

including coagulation, platelet aggregation, oxidant

stress, and subclinical inflammation?

� Is consumption of foods with added plant sterols/stanols

(2 g/day), as part of a healthy diet, associated with clinical

outcomes benefits? Given that large randomised out-

comes studies in low to moderate risk subjects are not

practically feasible, can sufficiently powered, randomised

controlled studies show significant effects of plant sterols

and/or stanols on morphological and functional (e.g.

endothelial function) surrogate markers of arterial

phenotype and/or atherosclerosis?

cost of these products is relevant as there appears to be a significantrelationship between socioeconomic level and the profile of con-sumption of food products [161,162]. Based on UK data, it is evidentthat the cost/kg of food products with added plant sterols can rangefrom 1.3-fold to up to 4-fold higher than that of their conventionalcounterparts (Table 2) [163]. Thus, cost may potentially constitute adeterrent to the regular purchase of these products, especiallyamong less affluent, higher-risk groups. Indeed, this is supported byrecent analyses from the Predi-Med study in Spain [164], whichsuggest that economic difficulties in Southern Europe may havehad a detrimental effect on adoption of favourable dietary behav-iours, including reduced adherence to the Mediterranean diet.

Further studies are needed to address unresolved questionshighlighted in this appraisal (see Box 5). Key priorities include (i)evaluation of the effects of plant sterols/stanols in patients withmetabolic syndrome; (ii) Mendelian randomisation studies toinvestigate the effects of plant sterols/stanols on clinical outcomes;and (iii) evaluation of long-term safety and effects on clinical out-comes in registries. Finally, although this EAS Consensus Panel didnot find evidence for any health risk associated with consumptionof these functional foods, the Panel recognises the lack of outcomesdata showing clinical benefit. Given practical constraints, and theinability to differentiate LDL-C lowering effects of concomitantpharmacotherapeutic and dietary approaches in polymedicatedpatients, health economic modelling (see Box 6) may offer afeasible approach to investigate whether wider use of these func-tional foods has the potential for healthcare savings due to reduc-tion in CVD prevalence. On the other hand, the EAS ConsensusPanel would clearly welcome, and applaud, efforts by the food in-dustry to plan and conduct a well-designed and adequately pow-ered study of the effects of plant sterol/stanol-supplemented foodson CVD outcomes.

Acknowledgements

We are indebted to Professor E. Bruckert for privilegedcommunication of unpublished findings in genetically-definedphytosterolaemic patients.

Jane Stock provided outstanding editorial support to theConsensus Panel.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360 357

Author contribution

European Atherosclerosis Society (EAS) Consensus Panel onPhytosterols

Writing committee: Helena Gylling (University of Helsinki,Finland), Lars Ellegård (Sahlgrenska Academy at University ofGothenburg, Sweden), Wendy Jessup (ANZAC Research Institute,Concord Hospital, Sydney, Australia), Peter J. Jones (RichardsonCentre for Functional Foods and Nutraceuticals, University ofManitoba, Winnipeg, Canada), Dieter Lütjohann (Institute of Clin-ical Chemistry and Pharmacology, University Clinics Bonn, Ger-many), Winfried Maerz (Synlab Academy, Synlab LLC, MannheimCenter of Laboratory Diagnostics; andMannheim Institute of PublicHealth, Social and Preventive Medicine, Medical Faculty Man-nheim, University of Heidelberg, Heidelberg, Germany; and ClinicalInstitute of Medical and Chemical Laboratory Diagnostics, MedicalUniversity of Graz, Austria), Luis Masana (University Hospital SantJoan, IISPV, CIBERDEM, Rovira and Virgili University, Reus, Spain),Jogchum Plat (Maastricht University, The Netherlands), GüntherSilbernagel (Swiss Cardiovascular Center, Inselspital, University ofBern, Switzerland), Bart Staels (University of Lille 2, Lille, France),Stephen Turley (UT Southwestern Medical Center, Dallas, USA).

Co-chairs: M. John Chapman (INSERM U939, Pitié-SalpetriereUniversity Hospital, Paris, France) and Henry N. Ginsberg (ColumbiaUniversity, New York, USA)

Other Panel members: Jan Borén (Sahlgrenska Center for Car-diovascular and Metabolic Research (CMR), University of Gothen-burg, Sweden), Alberico Catapano (University of Milan andMultimedica IRCSS Milano, Italy), Guy De Backer (Ghent UniversityHospital, Belgium), John Deanfield (National Centre for Cardiovas-cular Prevention and Outcomes, University College London, UK),Olivier S. Descamps (Hopital de Jolimont, Belgium), Petri T. Kovanen(Wihuri Research Institute, Helsinki, Finland), Gabriele Riccardi(Federico II University, Naples, Italy), and Lale Tokgözoglu (Hacet-tepe University, Ankara, Turkey).

The EAS Consensus Panel met twice in London, and the meet-ings were organised and chaired byMJC and HNG. The first meetingcritically reviewed the literature while the second meetingreviewed additional literature and scrutinized the first draft of theconsensus paper. Each Member of the Writing Committee draftedsections of themanuscript. All Panel members agreed to conceptionand design, contributed to interpretation of available data, sug-gested revisions for this document and all members approved thefinal document before submission.

Funding

This work including Consensus Panel meetings were supportedby unrestricted educational grants to the EAS from Unilever R & DVlaardingen B.V., The Netherlands; Danone Research, France; RaisioGroup, Finland; BASF SE, Germany and Johnson & Johnson Con-sumer Services EAME Ltd, UK. These companies were not present atthe Consensus Panel meetings, had no role in the design or contentof the Consensus Statement or in the nomination of the Panel, andhad no right to approve or disapprove the final document.

ST is fully supported by US Public Health Service GrantRO1HL09610.

Conflict of interest

In addition to unrestricted educational grants received by the EASreportedunderFunding, severalof theConsensusPanelmembershavereceived lecture honoraria, consultancy fees and/or research fundingfrom Abbott (HNG, LT), Aegerion (MJC,ALC), Amgen (MJC,LM), Astra

Zeneca (MJC,HNG,JB,OSD,LM,LT), Baxter (LE), Bayer (LT), Boehringer(HNG,LT), Bristol Myers Squibb (HNG), Danone (MJC,LM,OSD,JP,ST),Esteve (LM), Ferre (LM), Fresenius Kabi (LE), Genzyme (MJC,HNG),Hoffman-La Roche (MJC,LM), Kowa (ALC,MJC,HNG,LM,LT), Merck(MJC,HNG,JB, ALC,LM,OSD,ST,LT), Nestlé (LE), Novartis (HNG,LM,LT),Nutricia (LE), Pfizer (MJC,HNG,JB, OSD,LT), Raisio Nutrition Ltd(HGy,JP), Sanofi-Aventis/Regeneron (JB, OSD,HNG,LM,LT), Solvay(OSD), Unilever (DL,GS).

Appendix A. Supplementary material

Supplementary data related to this article can be found online athttp://dx.doi.org/10.1016/j.atherosclerosis.2013.11.043.

References

[1] Nichols M, Townsend N, Luengo-Fernandez R, et al. European cardiovasculardisease statistics 2012. Brussels/Sophia Antipolis: European Heart Network,European Society of Cardiology; 2012.

[2] Berry JD, Dyer A, Cai X, et al. Lifetime risks of cardiovascular disease. N Engl JMed 2012;366:321e9.

[3] Yusuf S, Hawken S, Ounpuu S, et al. Effect of potentially modifiable riskfactors associated with myocardial infarction in 52 countries (the INTER-HEART study): caseecontrol study. Lancet 2004;364:937e52.

[4] Baigent C, Keech A, Kearney PM, et al., Cholesterol Treatment Trialists’ (CTT)Collaborators. Efficacy and safety of cholesterol-lowering treatment: pro-spective meta-analysis of data from 90,056 participants in 14 randomisedtrials of statins. Lancet 2005;366:1267e78.

[5] Robinson JG, Smith B, Maheshwari N, Schrott H. Pleiotropic effects of statins:benefit beyond cholesterol reduction? A meta-regression analysis. J Am CollCardiol 2005;46:1855e62.

[6] Ference BA, Yoo W, Alesh I, et al. Effect of long-term exposure to lower low-density lipoprotein cholesterol beginning early in life on the risk of coronaryheart disease: a Mendelian randomization analysis. J Am Coll Cardiol2012;60:2631e9.

[7] Estruch R, Martínez-González MA, Corella D, et al. Effects of aMediterranean-style diet on cardiovascular risk factors: a randomized trial.Ann Intern Med 2006;145:1e11.

[8] Dietschy JM, Turley SD, Spady DK. Role of liver in the maintenance ofcholesterol and low density lipoprotein homeostasis in different animalspecies, including humans. J Lipid Res 1993;34:1637e59.

[9] Cooper AD. Hepatic uptake of chylomicron remnants. J Lipid Res 1997;38:2173e92.

[10] Davis Jr HR, Compton DS, Hoos L, Tetzloff G. Ezetimibe, a potent cholesterolabsorption inhibitor, inhibits the development of atherosclerosis in ApoEknockout mice. Arterioscler Thromb Vasc Biol 2001;21:2032e8.

[11] Chan DC, Watts GF, Gan SK, Ooi EM, Barrett PH. Effect of ezetimibe on he-patic fat, inflammatory markers, and apolipoprotein B-100 kinetics ininsulin-resistant obese subjects on a weight loss diet. Diabetes Care 2010;33:1134e9.

[12] Tremblay AJ, Lamarche B, Cohn JS, Hogue JC, Couture P. Effect of ezetimibe onthe in vivo kinetics of apoB-48 and apoB-100 in men with primary hyper-cholesterolemia. Arterioscler Thromb Vasc Biol 2006;26:1101e6.

[13] Klingberg S, Andersson H, Mulligan A, et al. Food sources of plant sterols inthe EPIC Norfolk population. Eur J Clin Nutr 2008;62:695e703.

[14] Valsta LM, Lemström A, Ovaskainen ML, et al. Estimation of plant sterol andcholesterol intake in Finland: quality of new values and their effect onintake. Br J Nutr 2004;92:671e8.

[15] Piironen V, Lampi AM. Occurrence and levels of phytosterols in foods. In:Dutta PC, editor. Phytosterols as functional food components and nutra-ceuticals. New York: Marcel Dekker, Inc; 2004. pp. 1e32.

[16] Vuoristo M, Miettinen TA. Absorption, metabolism, and serum concentra-tions of cholesterol in vegetarians: effects of cholesterol feeding. Am J ClinNutr 1994;59:1325e31.

[17] Ostlund Jr RE, McGill JB, Zeng C-M, et al. Gastro-intestinal absorption andplasma kinetics of soy D5-phytosterols and phytostanols in humans. Am JPhysiol Endocrinol Metab 2002;282:E911e6.

[18] Björkhem I, Boberg KM, Leitersdorf E. Inborn errors in bile acid biosynthesisand storage of sterols other than cholesterol. In: Scriver CR, Beaudet AL,Sly WS, Valle D, editors. The metabolic and molecular bases of inheriteddisease. 8th ed. New York: McGraw-Hill; 2001. pp. 2961e88.

[19] Fransen HP, de Jong N, Wolfs M, et al. Customary use of plant sterol and plantstanol enriched margarine is associated with changes in serum plant steroland stanol concentrations in humans. J Nutr 2007;137:1301e6.

[20] Berge KE, Tian H, Graf GA, et al. Accumulation of dietary cholesterol insitosterolemia caused by mutations in adjacent ABC transporters. Science2000;290:1771e5.

[21] Lee MH, Lu K, Hazard S, et al. Identification of a gene, ABCG5, important inthe regulation of dietary cholesterol absorption. Nat Genet 2001;27:79e83.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360358

[22] Salen G, von Bergmann K, Lütjohann D, et al., Multicenter SitosterolemiaStudy Group. Ezetimibe effectively reduces plasma plant sterols in patientswith sitosterolemia. Circulation 2004;109:966e71.

[23] Vanmierlo T, Popp J, Kölsch H, et al. The plant sterol brassicasterol as addi-tional CSF biomarker in Alzheimer’s disease. Acta Psychiatr Scand 2011;124:184e92.

[24] Salen G, Horak I, Rothkopf M, et al. Lethal atherosclerosis associated withabnormal plasma and tissue sterol composition in sitosterolemia with xan-thomatosis. J Lipid Res 1985;26:1126e33.

[25] Helske S, Miettinen T, Gylling H, et al. Accumulation of cholesterol precursorsand plant sterols in human stenotic aortic valves. J Lipid Res 2008;49:1511e8.

[26] Miettinen TA, Railo M, Lepäntalo M, Gylling H. Plant sterols in serum and inatherosclerotic plaques of patients undergoing carotid endarterectomy. J AmColl Cardiol 2005;45:1792e801.

[27] Miettinen TA, Nissinen M, Lepäntalo M, et al. Non-cholesterol sterols inserum and endarterectomized carotid arteries after a short-term plant stanoland sterol ester challenge. Nutr Metab Cardiovasc Dis 2011;21:182e8.

[28] Weingärtner O, Lütjohann D, Ji S, et al. Vascular effects of diet supplemen-tation with plant sterols. J Am Coll Cardiol 2008;51:1553e61.

[29] Mellies MJ, Ishikawa TT, Glueck CJ, Bove K, Morrison J. Phytosterols in aortictissue in adults and infants. J Lab Clin Med 1976;88:914e21.

[30] Jessup W, Herman A, Chapman MJ. Phytosterols in cardiovascular disease:innocuous dietary components, or accelerators of atherosclerosis? Fut Lip-idol 2008;3:301e10.

[31] Gylling H, Radhakrishnan R, Miettinen TA. Reduction of serum cholesterol inpostmenopausal women with previous myocardial infarction and choles-terol malabsorption induced by dietary sitostanol ester margarine: womenand dietary sitostanol. Circulation 1997;96:4226e31.

[32] Miettinen TA, Gylling H, Nissinen MJ. The role of serum non-cholesterolsterols as surrogate markers of absolute cholesterol synthesis and absorp-tion. Nutr Metab Cardiovasc Dis 2011;21:765e9.

[33] Descamps OS, De Sutter J, Guillaume M, Missault L. Where does the interplaybetween cholesterol absorption and synthesis in the context of statin and/orezetimibe treatment stand today? Atherosclerosis 2011;217:308e21.

[34] Rozner S, Garti N. The activity and absorption relationship of cholesteroland phytosterols. Colloids Surf A: Physicochem Eng Aspects 2006;282e283:435e56.

[35] Wang DQ-H. Regulation of intestinal cholesterol absorption. Annu RevPhysiol 2007;69:221e48.

[36] Turley SD. Role of Niemann-Pick C1-Like 1 (NPC1L1) in intestinal sterol ab-sorption. J Clin Lipidol 2008;2:S20e8.

[37] Nissinen M, Gylling H, Vuoristo M, Miettinen TA. Micellar distribution ofcholesterol and phytosterols after duodenal plant stanol ester infusion. Am JPhysiol Gastrointest Liver Physiol 2002;282:G1009e15.

[38] Nguyen TM, Sawyer JK, Kelley KL, Davis MA, Rudel LL. Cholesterol esterifi-cation by ACAT2 is essential for efficient intestinal cholesterol absorption:evidence from thoracic lymph duct cannulation. J Lipid Res 2012;53:95e104.

[39] Gylling HK, Hallikainen M, Vidgren H, Ågren J, Miettinen TA. Ester percent-ages of plant sterols and cholesterol in chylomicrons and VLDL of humanswith low and high sterol absorption. Atherosclerosis 2006;187:150e2.

[40] Calandra S, Tarugi P, Speedy HE, Dean AF, Bertolini S, Shoulders CC. Mech-anisms and genetic determinants regulating sterol absorption, circulatingLDL levels, and sterol elimination: implications for classification and diseaserisk. J Lipid Res 2011;52:1885e926.

[41] Lee S, Gershkovich P, Darlington J, Wasan K. Inhibition of cholesterol ab-sorption: targeting the intestine. Pharm Res 2012;29:3235e50.

[42] De Smet E, Mensink RP, Plat J. Effects of plant sterols and stanols on intestinalcholesterol metabolism: suggested mechanisms from past to present. MolNutr Food Res 2012;56:1058e72.

[43] Teupser D, Baber R, Ceglarek U, et al. Genetic regulation of serum phytosterollevels and risk of coronary artery disease. Circ Cardiovasc Genet 2010;3:331e9.

[44] Silbernagel G, Chapman MJ, Genser B, et al. High intestinal cholesterol ab-sorption is associated with risk alleles in ABCG8 and ABO and with cardio-vascular disease: evidence from the LURIC and YFS Cohorts and from a meta-analysis. J Am Coll Cardiol 2013;62:291e9.

[45] Andersson SW, Skinner J, Ellegård L, et al. Intake of plant sterols is inverselyrelated to serum cholesterol concentration in men and women in the EPICNorfolk population: a cross-sectional study. Eur J Clin Nutr 2004;58:1378e85.

[46] Klingberg S, Ellegård L, Johansson I, et al. Inverse relation between naturallyoccurring dietary plant sterols and serum cholesterol in northern Sweden.Am J Clin Nutr 2008;87:993e1002.

[47] Wang P, Chen YM, He LP, et al. Association of natural intake of dietary plantsterols with carotid intima-media thickness and blood lipids in Chineseadults: a cross-section study. PLoS ONE 2012;7:e32736.

[48] Lin X, Racette SB, Lefevre M, et al. The effects of phytosterols present innatural food matrices on cholesterol metabolism and LDL-cholesterol: acontrolled feeding trial. Eur J Clin Nutr 2010;64:1481e7.

[49] Racette SB, Lin X, Lefevre M, et al. Dose effects of dietary phytosterols oncholesterol metabolism: a controlled feeding study. Am J Clin Nutr 2010;91:32e8.

[50] Farquhar JW, Smith RE, Dempsey ME. The effect of beta sitosterol on theserum lipids of young men with arteriosclerotic heart disease. Circulation1956;14:77e82.

[51] Miettinen TA, Puska P, Gylling H, Vanhanen H, Vartiainen E. Reduction ofserum cholesterol with sitostanol-ester margarine in a mildly hypercholes-terolemic population. N Engl J Med 1995;333:1308e12.

[52] Katan MB, Grundy SM, Jones P, Law M, Miettinen T, Paoletti R, StresaWorkshop Participants. Efficacy and safety of plant stanols and sterols in themanagement of blood cholesterol levels. Mayo Clin Proc 2003;78:965e78.

[53] Demonty I, Ras RT, van der Knaap HC, et al. Continuous dose-responserelationship of the LDL-cholesterol-lowering effect of phytosterol intake.J Nutr 2009;139:271e84.

[54] Musa-Veloso K, Poon TH, Elliot JA, Chung C. A comparison of the LDL-cholsterol efficacy of plant stanols and plant sterols over a continuousrange: Results of a meta-analysis of randomized, placebo-controlled trials.Prostaglandins Leukot Essent Fatty Acids 2011;85:9e28.

[55] Demonty I, Ras RT, Trautwein EA. Comment on: “A comparison of the LDL-cholesterol lowering efficacy of plant stanols and plant sterols over acontinuous dose range: results of a meta-analysis of randomized, placebocontrolled trials” by Musa-Veloso K, Poon T H, Elliot JA, Chung C. Prosta-glandins Leukot Essent Fatty Acids 2011;85:9e28. Prostaglandins LeukotEssent Fatty Acids 2011;85:7e8.

[56] Reiner Z, Catapano AL, De Backer G, et al. ESC/EAS Guidelines for the man-agement of dyslipidaemias: the Task Force for the management of dyslipi-daemias of the European Society of Cardiology (ESC) and the EuropeanAtherosclerosis Society (EAS). Eur Heart J 2011;32:1769e818.

[57] Williams CL, Bollella MC, Strobino BA, Boccia L, Campanaro L. Plant stanolester and bran fiber in childhood: effects on lipids, stool weight and stoolfrequency in preschool children. J Am Coll Nutr 1999;18:572e81.

[58] Tammi A, Rönnemaa T, Miettinen TA, et al. Effects of gender, apolipoproteinE phenotype and cholesterol-lowering by plant stanol esters in children: TheSTRIP study. Acta Paediatr 2002;91:1155e62.

[59] Guardamagna O, Abello F, Baracco V, et al. Primary hyperlipidemias in chil-dren: effect of plant sterol supplementation on plasma lipids and markers ofcholesterol synthesis and absorption. Acta Diabetol 2011;48:127e33.

[60] Becker M, Staab D, von Bergmann K. Treatment of severe familial hyper-cholesterolemia in childhood with sitosterol and sitostanol. J Pediatr1993;122:292e6.

[61] Gylling H, Siimes MA, Miettinen TA. Sitostanol ester margarine in dietarytreatment of children with familial hypercholesterolemia. J Lipid Res1995;36:1807e12.

[62] Vuorio AF, Gylling H, Turtola H, Kontula K, Ketonen P, Miettinen TA. Stanolester margarine alone and with simvastatin lowers serum cholesterol infamilies with familial hypercholesterolemia caused by the FH-North Kareliamutation. Arterioscler Thromb Vasc Biol 2000;20:500e6.

[63] Amundsen AL, Ose L, Nenseter MS, Ntanios FY. Plant sterol ester-enrichedspread lowers plasma total and LDL cholesterol in children with familialhypercholesterolemia. Am J Clin Nutr 2002;76:338e44.

[64] de Jongh S, Vissers MN, Rol P, Bakker HD, Kastelein JJ, Stroes ES. Plant sterolslower LDL cholesterol without improving endothelial function in prepubertalchildren with familial hypercholesterolemia. J Inherit Metab Dis 2003;26:343e51.

[65] Ketomäki AM, Gylling H, Antikainen M, Siimes MA, Miettinen TA. Red celland plasma plant sterols are related during consumption of plant stanol andsterol ester spreads in children with hypercholesterolemia. J Pediatr2003;142:524e31.

[66] Jakulj L, Vissers MN, Rodenburg J, Wiegman A, Trip MD, Kastelein JJ. Plantstanols do not restore endothelial function in prepubertal children with fa-milial hypercholesterolemia despite reduction of low-density lipoproteincholesterol levels. J Pediatr 2006;148:495e500.

[67] Plat J, Mensink RP. Plant stanol esters lower serum triacylglycerol concen-trations via a reduced hepatic VLDL-1 production. Lipids 2009;44:1149e53.

[68] Sialvera TE, Pounis GD, Koutelidakis AE, et al. Phytosterols supplementationdecreases plasma small and dense LDL levels in metabolic syndrome patientson a westernized type diet. Nutr Metab Cardiovasc Dis 2012;22:843e8.

[69] Demonty I, Ras RT, van der Knaap HC, et al. The effect of plant sterols onserum triglyceride concentrations is dependent on baseline concentrations:a pooled analysis of 12 randomised controlled trials. Eur J Nutr 2013;52:153e60.

[70] Naumann E, Plat J, Kester ADM, Mensink RP. The baseline serum lipoproteinprofile is related to plant stanol induced changes in serum lipoproteincholesterol and triacylglycerol concentrations. J Am Coll Nutr 2008;27:117e26.

[71] Gylling H, Miettinen TA. Serum cholesterol and cholesterol and lipoproteinmetabolism in hypercholesterolaemic NIDDM patients before and duringsitostanol ester-margarine treatment. Diabetologia 1994;37:773e80.

[72] Plat J, Mensink RP. Vegetable oil based versus wood based stanol estermixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects. Atherosclerosis 2000;148:101e12.

[73] Mayne J, Dewpura T, Raymond A, et al. Plasma PCSK9 levels are significantlymodified by statins and fibrates in humans. Lipids Health Dis 2008;7:22.

[74] Kritchevsky D, Chen SC. Phytosterols e health benefits and potential con-cerns: a review. J Nutr Res 2005;25:413e28.

[75] Plat J, Beugels I, Gijbels MJ, de Winther MP, Mensink RP. Plant sterol or stanolesters retard lesion formation in LDL receptor-deficient mice independent ofchanges in serum plant sterols. J Lipid Res 2006;47:2762e71.

[76] Weingärtner O, Ulrich C, Lutjohann D, et al. Differential effects on inhibitionof cholesterol absorption by plant stanol and plant sterol esters in apoE�/�mice. Cardiovasc Res 2011;90:484e92.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360 359

[77] Hovenkamp E, Lourbakos A, Duchateau GS, Tareilus EW, Trautwein EA.Preferential efflux of phytosterols over cholesterol from macrophages. Lipids2007;42:1125e32.

[78] Bao L, Li Y, Deng SX, Landry D, Tabas I. Sitosterol-containing lipoproteinstrigger free sterol-induced caspase-independent death in ACAT-competentmacrophages. J Biol Chem 2006;281:33635e49.

[79] Tabas I, Feinmark SJ, Beatini N. The reactivity of desmosterol and othershellfish- and xanthomatosis-associated sterols in the macrophage sterolesterification reaction. J Clin Invest 1989;84:1713e21.

[80] Plat J, Nichols JA, Mensink RP. Plant sterols and stanols: effects on mixedmicellar composition and LXR (target gene) activation. J Lipid Res 2005;46:2468e76.

[81] Brauner R, Johannes C, Ploessl F, Bracher F, Lorenz RL. Phytosterols reducecholesterol absorption by inhibition of 27-hydroxycholesterol generation, liverX receptor a activation, and expression of the basolateral sterol exporter ATP-binding cassette A1 in Caco-2 enterocytes. J Nutr 2012;142:981e9.

[82] Kaneko E, Matsuda M, Yamada Y, Tachibana Y, Shimomura I, Makishima M.Induction of intestinal ATP-binding cassette transporters by a phytosterol-derived liver X receptor agonist. J Biol Chem 2003;278:36091e8.

[83] Yang C, Yu L, Li W, Xu F, Cohen JC, Hobbs HH. Disruption of cholesterolhomeostasis by plant sterols. J Clin Invest 2004;114:813e22.

[84] Sabeva NS, McPhaul CM, Li X, Cory TJ, Feola DJ, Graf GA. Phytosterolsdifferentially influence ABC transporter expression, cholesterol efflux andinflammatory cytokine secretion in macrophage foam cells. J Nutr Biochem2011;22:777e83.

[85] Calpe-Berdiel L, Escolá-Gil JC, Blanco-Vaca F. Are LXR-regulated genes a majormolecular target of plant sterols/stanols? Atherosclerosis 2007;195:210e1.

[86] Joseph SB, Castrillo A, Laffitte BA, Mangelsdorf DJ, Tontonoz P. Reciprocalregulation of inflammation and lipid metabolism by liver X receptors. NatMed 2003;9:213e9.

[87] Spann NJ, Garmire LX, McDonald JG, et al. Regulated accumulation of des-mosterol integrates macrophage lipid metabolism and inflammatory re-sponses. Cell 2012;151:138e52.

[88] Bouic PJ, Lamprecht JH. Plant sterols and sterolins: a review of their immune-modulating properties. Altern Med Rev 1999;4:170e7.

[89] Ding Y, Nguyen HT, Kim SI, Kim HW, Kim YH. The regulation of inflammatorycytokine secretion in macrophage cell line by the chemical constituents ofRhus sylvestris. Bioorg Med Chem Lett 2009;19:3607e10.

[90] Alappat L, Valerio M, Awad AB. Effect of vitamin D and b-sitosterol on im-mune function of macrophages. Int Immunopharmacol 2010;10:1390e6.

[91] Kurano M, Iso ON, Hara M, et al. Plant sterols increased IL-6 and TNF-asecretion from macrophages, but to a lesser extent than cholesterol.J Atheroscler Thromb 2011;18:373e83.

[92] Brüll F, Mensink RP, van den Hurk K, Duijvestijn A, Plat J. TLR2 activation isessential to induce a Th1 shift in human peripheral blood mononuclear cellsby plant stanols and plant sterols. J Biol Chem 2010;285:2951e8.

[93] Moreno JJ. Effect of olive oil minor components on oxidative stress andarachidonic acid mobilization and metabolism by macrophages RAW 264.7.Free Radic Biol Med 2003;35:1073e81.

[94] Vivancos M, Moreno JJ. Beta-sitosterol modulates antioxidant enzymeresponse in RAW 264.7 macrophages. Free Radic Biol Med 2005;39:91e7.

[95] Vivancos M, Moreno JJ. Effect of resveratrol, tyrosol and beta-sitosterol onoxidised low-density lipoprotein-stimulated oxidative stress, arachidonicacid release and prostaglandin E2 synthesis by RAW 264.7 macrophages. Br JNutr 2008;99:1199e207.

[96] Awad AB, Toczek J, Fink CS. Phytosterols decrease prostaglandin release incultured P388D1/MAB macrophages. Prostaglandins Leukot Essent FattyAcids 2004;70:511e20.

[97] Tedgui A, Mallat Z. Cytokines in atherosclerosis: pathogenic and regulatorypathways. Physiol Rev 2006;86:515e81.

[98] Raitakari OT, Salo P, Gylling H, Miettinen TA. Plant stanol ester consumptionand arterial elasticity and endothelial function. Br J Nutr 2008;100:603e8.

[99] Gylling H, Hallikainen M, Raitakari OT, et al. Long-term consumption of plantstanol and sterol esters, vascular function and genetic regulation. Br J Nutr2009;101:1688e95.

[100] Hallikainen M, Lyyra-Laitinen T, Laitinen T, et al. Endothelial function inhypercholesterolemic subjects: effects of plant stanol and sterol esters.Atherosclerosis 2006;188:425e32.

[101] Hallikainen M, Lyyra-Laitinen T, Laitinen T, Moilanen L, Miettinen TA,Gylling H. Effects of plant stanol esters on serum cholesterol concentrations,relative markers of cholesterol metabolism and endothelial function in type1 diabetes. Atherosclerosis 2008;199:432e9.

[102] Gylling H, Halonen J, Lindholm H, et al. The effects of plant stanol esterconsumption on arterial stiffness and endothelial function in adults: arandomised controlled clinical trial. BMC Cardiovasc Disord 2013;13:50.

[103] Raitakari OT, Salo P, Ahotupa M. Carotid artery compliance in users of plantstanol ester margarine. Eur J Clin Nutr 2008;62:218e24.

[104] Wang JJ, Liew G, Wong TY, et al. Retinal vascular calibre and the risk ofcoronary heart disease-related death. Heart 2006;92:1583e7.

[105] Kelly ER, Plat J, Mensink RP, Berendschot TTJM. Effects of long term plantsterol and -stanol consumption on the retinal vasculature: a randomizedcontrolled trial in statin users. Atherosclerosis 2011;214:225e30.

[106] Koz1owska-Wojciechowska M, Jastrzebska M, Naruszewicz M, Folty�nska A.Impact of margarine enriched with plant sterols on blood lipids, plateletfunction, and fibrinogen level in young men. Metabolism 2003;52:1373e8.

[107] De Jong A, Plat J, Bast A, Godschalk RW, Basu S, Mensink RP. Effects of plantsterol and stanol ester consumption on lipid metabolism, antioxidant statusand markers of oxidative stress, endothelial function and low-gradeinflammation in patients on current statin treatment. Eur J Clin Nutr2008;62:263e73.

[108] Othman RA, Moghadasian MH. Beyond cholesterol-lowering effects of plantsterols: clinical and experimental evidence of anti-inflammatory properties.Nutr Rev 2011l;69:371e82.

[109] Sudhop T, Gottwald BM, von Bergmann K. Serum plant sterols as a potentialrisk factor for coronary heart disease. Metabolism 2002;51:1519e21.

[110] Assmann G, Cullen P, Erbey J, Ramey DR, Kannenberg F, Schulte H. Plasmasitosterol elevations are associated with an increased incidence of coronaryevents in men: results of a nested case-control analysis of the ProspectiveCardiovascular Münster (PROCAM) study. Nutr Metab Cardiovasc Dis2006;16:13e21.

[111] Pinedo S, Vissers MN, von Bergmann K, et al. Plasma levels of plant sterolsand the risk of coronary artery disease: the prospective EPIC-Norfolk Pop-ulation Study. J Lipid Res 2007;48:139e44.

[112] Windler E, Zyriax BC, Kuipers F, Linseisen J, Boeing H. Association of plasmaphytosterol concentrations with incident coronary heart disease Data fromthe CORA study, a case-control study of coronary artery disease in women.Atherosclerosis 2009;203:284e90.

[113] Escurriol V, Cofán M, Moreno-Iribas C, et al. Phytosterol plasma concentra-tions and coronary heart disease in the prospective Spanish EPIC cohort.J Lipid Res 2010;51:618e24.

[114] Genser B, Silbernagel G, De Backer G, et al. Plant sterols and cardiovasculardisease: a systematic review and meta-analysis. Eur Heart J 2012;33:444e51.

[115] Tilvis RS, Valvanne JN, Strandberg TE, Miettinen TA. Prognostic significanceof serum cholesterol, lathosterol, and sitosterol in old age; a 17-year popu-lation study. Ann Med 2011;43:292e301.

[116] Weingärtner O, Pinsdorf T, Rogacev KS, et al. The relationships of markers ofcholesterol homeostasis with carotid intima-media thickness. PLoS ONE2010;5:e13467.

[117] Weingärtner O, Lütjohann D, Vanmierlo T, et al. Markers of enhancedcholesterol absorption are a strong predictor for cardiovascular diseases inpatients without diabetes mellitus. Chem Phys Lipids 2011;164:451e6.

[118] Fukushima M, Miura S, Mitsutake R, Fukushima T, Fukushima K, Saku K.Cholesterol metabolism in patients with hemodialysis in the presence orabsence of coronary artery disease. Circ J 2012;76:1980e6.

[119] IBC 50K CAD Consortium. Large-scale gene-centric analysis identifies novelvariants for coronary artery disease. PLoS Genet 2011;7:e1002260.

[120] Reilly MP, Li M, He J, et al., Myocardial Infarction Genetics Consortium,Wellcome Trust Case Control Consortium. Identification of ADAMTS7 as anovel locus for coronary atherosclerosis and association of ABO withmyocardial infarction in the presence of coronary atherosclerosis: twogenome-wide association studies. Lancet 2011;377:383e92.

[121] Schunkert H, König IR, Kathiresan S, et al., CARDIoGRAM Consortium. Largescale association analysis identifies 13 new susceptibility loci for coronaryartery disease. Nat Genet 2011;43:333e8.

[122] CARDIoGRAMplusC4D Consortium, Deloukas P, Kanoni S, Willenborg C, DIA-GRAM Consortium, CARDIOGENICS Consortium, MuTHER Consortium, Well-come Trust Case Control Consortium. Large-scale association analysis identifiesnew risk loci for coronary artery disease. Nat Genet 2013;45:25e33.

[123] Strandberg TE, Tilvis RS, Pitkala KH, Miettinen TA. Cholesterol and glucosemetabolism and recurrent cardiovascular events among the elderly: a pro-spective study. J Am Coll Cardiol 2006;48:708e14.

[124] Silbernagel G, Fauler G, Renner W, et al. The relationships of cholesterolmetabolism and plasma plant sterols with the severity of coronary arterydisease. J Lipid Res 2009;50:334e41.

[125] Matthan NR, Pencina M, LaRocque JM, et al. Alterations in cholesterol ab-sorption/synthesis markers characterize Framingham offspring study par-ticipants with CHD. J Lipid Res 2009;50:1927e35.

[126] Silbernagel G, Fauler G, Hoffmann MM, et al. The associations of cholesterolmetabolism and plasma plant sterols with all-cause and cardiovascularmortality. J Lipid Res 2010;51:2384e93.

[127] Smith NL, Chen MH, Dehghan A, et al., Wellcome Trust Case Control Con-sortium. Novel associations of multiple genetic loci with plasma levels offactor VII, factor VIII, and von Willebrand factor: the CHARGE (Cohorts forHeart and Aging Research in Genome Epidemiology) Consortium. Circulation2010;121:1382e92.

[128] Kiechl S, Paré G, Barbalic M, et al. Association of variation at the ABO locuswith circulating levels of soluble intercellular adhesion molecule-1, solubleP-selectin, and soluble E-selectin: a meta-analysis. Circ Cardiovasc Genet2011;4:681e6.

[129] Karakas M, Baumert J, Kleber ME, et al. A variant in the abo gene explains thevariation in soluble e-selectin levels-results from dense genotyping in twoindependent populations. PLoS ONE 2012;7:e51441.

[130] Fassbender K, Lütjohann D, Dik MG, et al. Moderately elevated plant sterollevels are associated with reduced cardiovascular risk-the LASA study.Atherosclerosis 2008;196:283e8.

[131] Buchwald H, Varco RL, Boen JR, et al. Effective lipid modification by partialileal bypass reduced long-term coronary heart disease mortality andmorbidity: five-year posttrial follow-up report from the POSCH. Program onthe Surgical Control of the Hyperlipidemias. Arch Intern Med 1998;158:1253e61.

H. Gylling et al. / Atherosclerosis 232 (2014) 346e360360

[132] Brown L, Rosner B, Willett WW, Sacks FM. Cholesterol-lowering effects ofdietary fiber: a meta-analysis. Am J Clin Nutr 1999;69:30e42.

[133] Pereira MA, O’Reilly E, Augustsson K, et al. Dietary fiber and risk of coronaryheart disease: a pooled analysis of cohort studies. Arch Intern Med2004;164:370e6.

[134] Willems JI, Blommaert MAE, Trautwein EA. Results from a post-launchmonitoring survey on consumer purchases of foods with added phytos-terols in five European countries. Food Chem Toxicol 2013;62:48e53.

[135] Lea LJ, Hepburn PA. Safety evaluation of phytosterol-esters. Part 9: Results ofa European post-launch monitoring programme. Food Chem Toxicol2006;44:1213e22.

[136] EFSA Panel on Food Additives and Nutrient Sources added to Food (ANS).Scientific opinion on the safety of stigmasterol-rich plant sterols as foodadditive. EFSA J 2012;10:2659.

[137] Hendriks HF, Brink EJ, Meijer GW, Princen HM, Ntanios FY. Safety of long-term consumption of plant sterol esters-enriched spread. Eur J Clin Nutr2003;57:681e92.

[138] de Jong A, Plat J, Lütjohann D, Mensink RP. Effects of long-term plant sterolor stanol ester consumption on lipid and lipoprotein metabolism in subjectson statin treatment. Br J Nutr 2008;100:937e41.

[139] Noakes M, Clifton P, Ntanios F, Shrapnel W, Record I, McInerney J. An in-crease in dietary carotenoids when consuming plant sterols or stanols iseffective in maintaining plasma carotenoid concentrations. Am J Clin Nutr2002;75:79e86.

[140] Tuomilehto J, Tikkanen MJ, Högström P, et al. Safety assessment of commonfoods enriched with natural nonesterified plant sterols. Eur J Clin Nutr2009;63:684e91.

[141] Plat J, Mensink RP. Effects of diets enriched with two different plant stanolester mixtures on plasma ubiquinol-10 and fat-soluble antioxidant concen-trations. Metabolism 2001;50:520e9.

[142] Baskar AA, Ignacimuthu S, Paulraj GM, Al Numair KS. Chemopreventivepotential of beta-sitosterol in experimental colon cancer model e an in vitroand in vivo study. BMC Compl Altern Med 2010;10:24.

[143] Lea LJ, Hepburn PA, Wolfreys AM, Baldrick P. Safety evaluation of phytosterolesters. Part 8. Lack of genotoxicity and subchronic toxicity with phytosteroloxides. Food Chem Toxicol 2004;42:771e83.

[144] Wolfreys AM, Hepburn PA. Safety evaluation of phytosterol esters. Part 7.Assessment of mutagenic activity of phytosterols, phytosterol esters and thecholesterol derivative, 4-cholesten-3-one. Food ChemToxicol 2002;40:461e70.

[145] Woyengo TA, Ramprasath VR, Jones PJ. Anticancer effects of phytosterols. EurJ Clin Nutr 2009;63:813e20.

[146] De Stefani E, Boffetta P, Ronco AL, et al. Plant sterols and risk of stomachcancer: a case-control study in Uruguay. Nutr Cancer 2000;37:140e4.

[147] Mendilaharsu M, De Stefani E, Deneo-Pellegrini H, Carzoglio J, Ronco A.Phytosterols and risk of lung cancer: a case-control study in Uruguay. LungCancer 1998;21:37e45.

[148] Blair SN, Capuzzi DM, Gottlieb SO, Nguyen T, Morgan JM, Cater NB. Incre-mental reduction of serum total cholesterol and low-density lipoproteincholesterol with the addition of plant stanol ester-containing spread tostatin therapy. Am J Cardiol 2000;86:46e52.

[149] Neil HA, Meijer GW, Roe LS. Randomised controlled trial of use by hyper-cholesterolaemic patients of a vegetable oil sterol-enriched fat spread.Atherosclerosis 2001;156:329e37.

[150] Castro Cabezas M, de Vries JH, Van Oostrom AJ, Iestra J, van Staveren WA.Effects of a stanol-enriched diet on plasma cholesterol and triglycerides inpatients treated with statins. J Am Diet Assoc 2006;106:1564e9.

[151] Simons LA. Additive effect of plant sterol-ester margarine and cerivastatin inlowering low-density lipoprotein cholesterol in primary hypercholesterole-mia. Am J Cardiol 2002;90:737e40.

[152] Gylling H, Miettinen TA. Effects of inhibiting cholesterol absorption andsynthesis on cholesterol and lipoprotein metabolism in hypercholesterol-emic non-insulin-dependent diabetic men. J Lipid Res 1996;37:1776e85.

[153] Lin X, Racette SB, Lefevre M, et al. Combined effects of ezetimibe and phy-tosterols on cholesterol metabolism: a randomized, controlled feeding studyin humans. Circulation 2011;124:596e601.

[154] Becker M, Staab D, Von Bergman K. Long-term treatment of severe familialhypercholesterolemia in children: effect of sitosterol and bezafibrate. Pedi-atrics 1992;89:138e42.

[155] Nigon F, Serfaty-Lacrosnière C, Beucler I, et al. Plant sterol-enriched margarinelowers plasma LDL in hyperlipidemic subjects with low cholesterol intake:effect of fibrate treatment. Clin Chem Lab Med 2001;39:634e40.

[156] Micallef MA, Garg ML. Beyond blood lipids: phytosterols, statins and omega-3 polyunsaturated fatty acid therapy for hyperlipidemia. J Nutr Biochem2009;20:927e39.

[157] O’Neill FH, Brynes A, Mandeno R, et al. Comparison of the effects of dietaryplant sterol and stanol esters on lipid metabolism. Nutr Metab CardiovascDis 2004;14:133e42.

[158] Relas H, Gylling H, Miettinen TA. Effect of stanol ester on postabsorptivesqualene and retinyl palmitate. Metabolism 2000;49:473e8.

[159] Gylling H, Hallikainen M, Simonen P, Miettinen HE, Nissinen MJ,Miettinen TA. Serum and lipoprotein sitostanol and non-cholesterol sterolsafter an acute dose of plant stanol ester on its long-term consumption. Eur JNutr 2012;56:663e70.

[160] Demonty I, Chan YM, Pelled D, Jones PJ. Fish-oil esters of plant sterolsimprove the lipid profile of dyslipidemic subjects more than do fish-oil orsunflower oil esters of plant sterols. Am J Clin Nutr 2006;84:1534e42.

[161] Darmon N, Drewnowski A. Does social class predict diet quality? Am J ClinNutr 2008;87:1107e17.

[162] Bonaccio M, Bonanni AE, Di Castelnuovo A, et al. Low income is associatedwith poor adherence to a Mediterranean diet and a higher prevalence ofobesity: cross-sectional results from the Moli-sani study. BMJ Open 2012;2:e001685. http://dx.doi.org/10.1136/bmjopen-2012-001685.

[163] European Food Safety Authority. A report from the data collection andexposure unit in response to a request from the European Commission. EFSAJ 2008;133:1e21. http://www.efsa.europa.eu/en/scdocs/doc/datex_report_ej133_phytosterols_en.pdf [accessed 04.07.13].

[164] Bes-Rastrollo M. Costs of Mediterranean and Western dietary patterns andtheir relationship with prospective weight change. EuroPRevent 2013 [ab-stract 610].